Zinc supplementation boosts the stress response in the elderly: Hsp70 status is linked to zinc availability in peripheral lymphocytes.

Chaperones and zinc are indispensable for proper immune function. All the zinc status, the immune function and the stress response decline during aging. Here we studied the effect of nutritional zinc and zinc homeostasis on the stress response in healthy old subjects recruited during the ZincAge European Union project that either underwent or not a 48-day zinc supplementation. Inducible Hsp70 levels were determined at basal conditions as well as after heat shock in the CD3+ and CD3- subset of lymphocytes by a two-color FACS analysis. Short term zinc supplementation resulted in a marked increase in both basal as well as stress-induced Hsp70 levels in lymphocytes from healthy elderly donors with a higher impact on CD3+ cells. Heat inducibility showed a strong correlation with basal Hsp70 level, and both basal as well as stress-induced Hsp70 highly correlated with intracellular zinc availability. In conclusion, short term oral supplementation with zinc safely and efficiently induces the stress response in lymphocytes of old donors. The stress response may be a candidate pathway connecting zinc deficiency with aging and immunosenescence. Thus, proper dietary zinc intake may emerge as a chaperone inducer and an anti-aging mechanism in the immune system.

GM-CSF DNA: an adjuvant for higher avidity IgG, rectal IgA, and increased protection against the acute phase of a SHIV-89.6P challenge by a DNA/MVA immunodeficiency virus vaccine.

Single intradermal or intramuscular inoculations of GM-CSF DNA with the DNA prime for a simian-human immunodeficiency virus (SHIV)-89.6 vaccine, which consists of DNA priming followed by modified vaccinia Ankara (MVA) boosting, increased protection of both the blood and intestines against the acute phase of an intrarectal SHIV-89.6P challenge. GM-CSF appeared to contribute to protection by enhancing two antibody responses: the avidity maturation of anti-Env IgG in blood (p=or<0.01) and the presence of long lasting anti-viral IgA in rectal secretions (p<0.01). The avidity of anti-Env IgG showed strong correlations with protection both pre and post challenge. Animals with the highest avidity anti-Env Ab had 1000-fold reductions in peak viremia over those with the lowest avidity anti-Env Ab. The enhanced IgA response was associated with the best protection, but did not achieve significance.

Novel treatment in peritoneal adhesion prevention: protection by polypeptides.

OBJECTIVE: To evaluate a novel antiadhesive polypeptide complex containing a combination of poly-L-glutamate and poly-L-lysine in order to study its effectiveness and mechanisms in the prevention of postoperative abdominal adhesions in mice. MATERIAL AND METHODS: The length of peritoneal adhesions was measured and expressed in percentage of the wound length in a standardized peritoneal injury model and evaluated 7 days and 4 weeks after adhesion induction. The test compound was administered intraperitoneally following surgery. Peritoneal swabs, including the wound area, were stained in order to determine the peritoneal location and clearance of the polypeptides. Electron microscopy was performed to analyze the wound surface and the ultra-structural changes of the phagocytes in cell culture. Moreover, flow cytometry was used to evaluate the effect on macrophage phagocytic function. RESULTS: The poly-L-lysine and poly-L-glutamate combination significantly decreased peritoneal adhesions both at 7 days' (p < 0.001) and 4 weeks' (p < or = 0.001) follow-up. From the first day, the compound was found in the wound, after which this was gradually rebuilt, and covered with mesothelial cells. The macrophages phagocytosed the test compound particles, resulting in significant cell growth, and large phagocytic vacuoles. CONCLUSIONS: The intraperitoneal administration of poly-L-lysine and poly-L-glutamate resulted in a significant decrease in experimental postoperative peritoneal adhesions.

Comparative studies on mucosal and intravenous transmission of simian immunodeficiency virus (SIVsm): evolution of coreceptor use varies with pathogenic outcome.

Coreceptor usage of isolates from 30 cynomolgus macaques infected intrarectally (n=22) or intravenously (n=8) with simian immunodeficiency virus of sooty mangabey origin (SIVsm) was evaluated in U87.CD4 and GHOST(3) cell lines. Based on progression rate, the animals were divided into progressors (18 animals), slow progressors (five animals) and long-term non-progressors (seven animals). There was no difference in how many or which coreceptors were used according to route of infection. All isolates but one used CCR5 for cell entry, and CCR5 was also the major coreceptor in 70 out of 105 isolates tested. In general, early isolates were multitropic, using CCR5, CXCR6 and/or gpr15. Interestingly, CXCR4-using viruses could be isolated on human peripheral blood mononuclear cells (PBMCs), but not on cynomolgus macaque PBMCs, suggesting that human PBMCs select for variants with CXCR4 use. Even though CXCR4-using SIV isolates have been reported rarely, we could recover CXCR4-using viruses from 13 monkeys. CXCR4 use either appeared early during the acute phase of infection and disappeared later or only appeared late in infection during immunodeficiency. Surprisingly, one late isolate from a progressor monkey did not use CCR5 at all and used the CXCR4 receptor with high efficiency. The ability to use many different receptors decreased over time in long-term non-progressor monkeys, whilst the majority of progressor monkeys showed broadening of coreceptor use, stable coreceptor use or fluctuation between the different coreceptor-usage patterns. The results indicate that, in the infected host, evolution of SIV coreceptor usage occurs, involving changes in the mode of coreceptor use.

Prevention of postoperative peritoneal adhesions: effects of lysozyme, polylysine and polyglutamate versus hyaluronic acid.

OBJECTIVE: Intraperitoneal adhesions are an important cause of postoperative intestinal obstruction, abdominal discomfort and infertility. In the present study we hypothesized that a combination of polypeptides with different surface properties, resulting in fine disperse low-soluble complexes, could be of benefit in the prevention of abdominal adhesions. MATERIAL AND METHODS: Various polypeptides including lysozyme, polyglutamate, polylysine and combinations of all three were evaluated as compared to hyaluronic acid. A standard wound on the parietal peritoneum in mice was used and the evaluated agents were administered immediately postoperatively. The extent of peritoneal adhesions to the injured area was measured and expressed as a percentage of the wound length as evaluated after 7 days. Flow cytometry was performed to evaluate the effect on peritoneal macrophage survival and phagocytic function and the Pick test was used to determine peroxide production in order to estimate toxicity and potential impairment of macrophage function caused by the chemicals. RESULTS: Significant differences were seen among the treatment groups (p<0.001). Both polyglutamate and lysozyme, and polyglutamate together with polylysine significantly decreased adhesion formation as compared to hyaluronic acid. The polylysine-polyglutamate combination was still visible macroscopically on the peritoneal surface after 1 week, though not after 1 month. The polyglutamate-lysozyme mixture was less effective than these individual components alone. The chemicals did not show any toxic effects or altered function in macrophage cell culture. CONCLUSIONS: Lysozyme, polyglutamate and, most effectively, a polyglutamate-polylysine combination significantly decreased experimental abdominal adhesion formation. A strong mechanical connection to the wound and prolonged attendance in the surface were noted. Peritoneal phagocyte function did not seem to be influenced by the chemicals.

Quantitative evaluation of HIV and SIV co-receptor use with GHOST(3) cell assay.

An assay has been established for quantitative evaluation of lentivirus coreceptor use with the help of GHOST(3) cells. GHOST(3) cells were derived from the human osteosarcoma cell line, HOS, and have been engineered to stably express CD4 and one or another of the chemokine receptors CCR3, CCR5, CXCR4, CXCR6/STRL33/Bonzo, or the orphan receptor GPR15/BOB. The indicator cell line carries the HIV-2 long terminal repeat-driven green fluorescence protein (GFP) gene, which becomes activated upon infection with HIV or SIV. Viral entry is followed by Tat activation of transcription and GFP becomes expressed. Infected cells can be detected as early as 2 or 3 d after infection by simple fluorescence microscopic observation. The simplicity of the GHOST(3) cell system makes it particularly suitable for screening of a large number of isolates. In addition, the efficiency of co-receptor use can be accurately quantitated with flow cytometric analysis. Thus, the most efficiently used co-receptor of multitropic isolates can be determined. It is also possible to sensitively determine co-receptor switch of sequential isolates from the same individual.

Primate models for human immunodeficiency virus infection. Evolution of receptor use during pathogenesis.

Animal models greatly facilitate understanding of transmission, pathogenesis and immune responses in HIV and SIV infection and provide models for studies on the effect of candidate drugs or vaccines. However, there are several aspects that one should consider when drawing conclusions from results obtained from animal models. First, the genetic relationship of primate lentiviruses cannot be disregarded because it is known that HIV-1 is more closely related to SIV of chimpanzee origin (SIVcpz) than to SIV from sooty mangabey (SIVsm) origin. Nevertheless, SIVsm and SIVmac are the ones most often used as model systems. Second, there are differences in the biological properties, like CXCR4 use and CD4-independent coreceptor use, of HIV and SIV. These differences might be relevant in virus transmission, pathogenesis and in evoking immune responses. Third, in vivo and in vitro selection may influence the results. Neutralizing antibodies may play a role in selection of variant viruses since neutralization sensitive, CD4-independent SIVsm variants seemed to be suppressed in animals that mounted a neutralizing antibody response. It is tempting to speculate that neutralizing antibodies shape the SIV/HIV infection by selecting variants with a more "closed" envelope conformation with consequences for both receptor binding and neutralization sensitivity. The SIV/monkey model, although it has important advantages, may not answer all questions asked about HIV-1 infection in human.

Evolution of coreceptor use and CD4-independence in envelope clones derived from SIVsm-infected macaques.

Coreceptor use of HIV can evolve during infection. We previously examined coreceptor use of related SIVsm inoculum viruses and sequential reisolates from cynomolgus macaques. These viruses exhibited broad coreceptor specificities and, generally, CCR5 use remained efficient and stable, while alternative coreceptor use decreased longitudinally. Here we demonstrate that individual envelopes (Envs) from inoculum and reisolate viruses fuse via a range of coreceptors, including CCR5, CCR8, CXCR6, GPR15, GPR1, and APJ. On the whole, coreceptor use of Envs from sequential reisolates recapitulated that of reisolate viruses, thus CCR5 use remained stable while alternative coreceptor use tended to decrease over time. Rhesus CCR5, GPR15, and CXCR6 supported fusion to a similar extent as their human counterparts. Additionally, a number of Envs mediated CD4-independent fusion via CCR5 and GPR15. Envs from different inoculum viruses exhibited distinct dependencies on CD4 for fusion via CCR5, ranging from strictly CD4-dependent to efficiently CD4-independent. Early reisolates from macaques infected with CD4-independent inoculums maintained or evolved Envs with a broad range of CD4-independence. CD4-independence became less variable/efficient in late reisolates from macaques that developed neutralizing antibodies. Infection with a CD4-dependent virus resulted in evolution of CD4-independent Envs in late reisolates. While CD4 independence can potentially broaden tropism in vivo, CD4-independent viruses are particularly sensitive to neutralizing antibodies. Therefore, interplay between receptor tropism and neutralization may shape viral evolution and SIV pathogenesis.